Experimental Studies Of Gene Therapy After Transient Focal Cerebral Ischemia In Rats

Objective: To investigate whether neuroprotective effect can be achieved by liposome mediated TGF- β1 gene transfer to rat brain after transient focal cerebral ischemia and whether transplantation of neural stem cells can ameliorate memory dificits in neonatal hypoxic-ischemic brain injury in rats.Methods: Transient focal cerebral ischemia in a rat model was produced by middle cerebral artery occlusion (MCAO) for 1 hour. Reliable behavioral parameters were employed to detect successful occlusion of the middle cerebral artery. The plasmid contained LacZ marker gene, complexed to cationic liposomes, were injected directly into the cerebral fluid (CSF) of one group of control animals (n=16) immediately after MCAO. The other group of animals (n=16) was injected with liposome-TGF-β1 complexes. Five days after transfection, behavioral tests were employed to evaluate the neurologic deficits. Expression of p-galactosidase and TGF-β1 gene products were detected by histochemistry, reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. Infarct volume and TUNEL-positve cells were counted. Semi-quantitative RT-PCR was used to examine the difference of caspase-3 mRNA between the two groups. The cells from the embryonic rat cortices were mechanically dissociated. N2 medium was adapted to culture the cells. EGF and bFGF were added to expand the cells. Neural stem cells and differentiated cells were identified by immunocytochemistry. Newborn rats were sustained hypoxic-ischemic brain injury. One week later, animals received neural stem cells transplants (operate group=16) or PBS transplants (control group=8). At 6-8weeks posttransplantation, animals' memory ability were observed by Y-maze test. Then, animals were sarcificed and their brains histologically examined.Results Liposome-mediated gene transfer of LacZ and TGF- β1 into the ischemic brain provided effective expression of transgene. Compared with the control groups, the neurologic function of the rats injected with liposome-TGF-β1complexes was significantly improved (P<0.01) ; infarct volume was smaller significantly (P<0.01) ; TUNEL-positive cells decreased significantly (P<0.01) and the expression of caspase-3 mRNA also decreased significantly. Neural stem cells from embryonic brains have been successfully cultured and they formed typical neurospheres in suspension, and the majorities of the cells expressed nestin, which was the marker for neural stem cells. We show that animals that received neural stem cells grafts 7 days following the hypoxic-ischemic brain injury performed significantly better as adults on measures of memory ability, the Y-maze test, than did control animals that received sham transplants after the hypoxic-ischemic injury. Survivals of transplants were demonstrated in animals.Conclusion Liposome-mediated gene transfer into the ischemic brain provided effective expression of transgene after transient focal cerebral ischemia. Neuroprotective effect can be achived by liposome mediated TGF-β1 gene tranfer which resulted to the inhibition of the activity of caspase-3. Gene transfer mediated by liposome may be a promising approach for treament of cerebral ischemia. Neural stem cells could be cultured from embryonic brains, and they formed the typical neurospheres in suspention in vitro. Transplantation of neural stem cells can ameliorate memory dificits following neonatal IH brain injury in rats.